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CRISPR/Cas9 in Genome
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Editing and Beyond
Haifeng Wang,1 Marie La Russa,1,2 and Lei S. Qi1,3,4
Annu. Rev. Biochem. 2016.85:227-264. Downloaded from www.annualreviews.org
1
Department of Bioengineering, Stanford University, Stanford, California 94305;
email: hfgwang@stanford.edu, mlarussa@stanford.edu, stanley.qi@stanford.edu
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2
Biomedical Sciences Graduate Program, University of California, San Francisco,
California 94158
3
Department of Chemical and Systems Biology, Stanford University, Stanford, California 94305
4
Chemistry, Engineering and Medicine for Human Health (ChEM–H), Stanford University,
Stanford, California 94305
227
BI85CH10-Qi ARI 9 May 2016 9:29
Contents
INTRODUCTION: TOOLS FOR PROGRAMMABLE GENOME EDITING,
TARGETING, AND REGULATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
CRISPR/CAS9: A GIFT FROM MOTHER NATURE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
CRISPR: An Adaptive Immune Mechanism in Bacteria and Archaea. . . . . . . . . . . . . . . 231
Repurposing Cas9 for Sequence-Specific Genomic Targeting . . . . . . . . . . . . . . . . . . . . . 232
Diversity of Cas9 Orthologs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
Engineered Variants of the Cas9 Nuclease Domains: Nickase Cas9 (nCas9)
and Nuclease-Deactivated Cas9 (dCas9) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Other RNA-Guided Endonucleases in the CRISPR Systems . . . . . . . . . . . . . . . . . . . . . . 233
STRUCTURE OF Cas9 AND PROPOSED WORKING MODEL FOR GUIDE
RNA BINDING AND TARGET DNA CLEAVAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
Annu. Rev. Biochem. 2016.85:227-264. Downloaded from www.annualreviews.org
Since the advent of the central dogma of molecular biology, scientists have endeavored to develop
new technologies to modify or manipulate the genome. Precise editing and regulation of genomic
information is essential to understanding the function of a given gene. During the past decade,
technological breakthroughs have made genome editing and regulation significantly easier. One
recent technology has adapted the CRISPR (clustered regularly interspaced short palindromic
repeats)/Cas (CRISPR-associated protein) bacterial immune system as a simple, RNA-guided
platform for highly efficient and specific genome editing and regulation in diverse organisms,
thus creating revolutionary tools for biomedical research and new possibilities for treating genetic
disorders (1–14).
In general, the precise editing or regulation of genomic information at the DNA level requires
the action of a molecular machine composed of two major parts: a DNA-binding domain that
mediates sequence-specific DNA recognition and binding, and an effector domain that enables
DNA cleavage or regulates transcription near the binding site. Creating a double-stranded break
(DSB) by using a sequence-specific endonuclease can stimulate the DNA repair pathway and
greatly increase the rate of gene modification at the desired sequence (15–20). Thus, nuclease-
mediated approaches have been extensively explored for site-specific gene editing. Meganucleases,
or homing nucleases, are among the first classes of nucleases that were engineered to target specific
genomic sites for gene editing purposes (15, 16, 21). Meganucleases are a group of nucleases that
recognize long nucleotide sequences and induce a DSB at their targeted site. The long recognition
sequence of meganucleases may occur only once within a genome, thereby facilitating its use
for site-specific genome editing. Meganucleases can be reengineered to target novel sequences
through strategies such as protein engineering, structure-based design, and molecular evolution,
although the procedure is usually labor intensive (20–22).
Other examples of programmable genome editing machines include zinc-finger nucleases
(ZFNs) (23–25) and transcription activator-like effector nucleases (TALENs) (26–28), in which
the DNA-binding domains of transcription factors have been fused with the nuclease domain of
the restriction enzyme FokI, an obligate dimer (Figure 1a). When targeted to paired adjacent
sequences, the FokI domains of these programmable, site-specific nucleases form a dimer that ac-
tivates the nuclease activity, thus creating a DSB near their binding sites (Figure 1a). Researchers
can exploit the cell’s endogenous DNA repair pathways to create mutations at the desired DSB
sites. However, because these tools function through protein–DNA interactions, targeting to a
new site requires engineering and cloning a new protein, which precludes ZFNs and TALENs
from being used for high-throughput applications.
a
Nucleases based on protein–DNA interactions
FokI FokI
FokI FokI
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RNA-directed nucleases
Sp Cas9 Sa Cas9
Cas9
REC REC
REC sgRNA
3' sgRNA
20nt 5' HNH
b Cas9
sgRNA
Silencing mutation
Homology-directed repair (HDR)
Double-stranded break (DSB)
Targeted sequence replacement
dCas9-FokI (mutations, gene correction,
gene knock-in, etc.)
Donor DNA
FokI
FokI
sequence-specific gene editing, as well as other applications. Its natural endonuclease activity has
been co-opted for sequence-specific editing of the genome in a wide range of organisms, including
bacteria (31), fungi (32), plants (33, 34), and animals (5–7, 9, 10, 35, 36). To enable sequence-
specific genomic regulation, nuclease-deactivated Cas9 (dCas9) has been engineered, and can
be fused to a variety of effectors, such as transcriptional activators, repressors, and epigenetic
modifiers (37–41).
In addition to applications in genome editing and regulation, DNA-binding proteins, such as
ZFs, TALEs, and dCas9, have been fused to fluorescent proteins (FPs) to allow direct imaging of
genomic loci in living cells (42–47). Additionally, dCas9 has also been used for studying proteins
that interact with specific loci (48, 49), and it may potentially be used to target RNA (50, 51). In
this review, we describe the working mechanism of Cas9 based on the findings of structural and
biochemical studies. We focus on the applications of CRISPR/Cas9 in genomic editing, regulation,
and imaging in mammalian cells, highlighting the power of this novel system in biological research.
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←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
−
Figure 1
CRISPR-associated protein 9 (Cas9)-mediated sequence-specific genomic editing. (a) Comparison of programmable sequence-specific
genome editing nucleases. (Top) Zinc-finger nucleases (ZFNs) and transcriptional activator-like effector nucleases (TALENs) are
engineered by fusing ZF or TALE DNA-binding domains to the FokI nuclease domains (23–28). They recognize their targeted sites by
sequence-specific protein–DNA interactions, and a pair of ZFNs or TALENs cleaves adjacent sequences of the DNA to create a pair of
nicks on complementary strands, leading to a double-stranded break (DSB). (Bottom) Cas9 is a naturally evolved, RNA-guided nuclease.
It recognizes its target DNA through approximately 20 nucleotide (nt) base-pairing interaction between a single guide RNA (sgRNA)
and its targeted DNA strand. Cas9 also interacts with the protospacer-adjacent motif (PAM) of its DNA target through its
PAM-interacting (PI) domain at its C terminus. Cas9 uses its two nuclease domains (HNH and RuvC) to cleave the double-stranded
DNA, creating a DSB. The HNH, RuvC, and PI domains, as well as an evolutionarily divergent wedge domain (WED), all reside in
the Cas9 nuclease (NUC) lobe. The recognition (REC) lobe of Cas9 contains other regions that interact with the sgRNA–DNA
duplex. (Bottom right) Crystal structures of Sp Cas9 and Sa Cas9. Crystal structures of Streptococcus pyogenes Cas9 (Sp Cas9; Protein Data
Bank number 4UN3, 1368 AA) (84) and Staphylococcus aureus Cas9 (Sa Cas9 Protein Data Bank number 5CZZ, 1053 AA) (70) were
obtained from RCSB Protein Data Bank (http://www.rcsb.org/pdb/), compared using PyMOL (PyMOL Molecular Graphics System,
Version 1.3, Schrödinger LLC, https://www.pymol.org/), and domains are annotated according to References 70, 81, 83, 84. The
orientation of the target DNA strand is also shown. (b) Cas9 in genomic editing. The DSB generated by Cas9 activates the
nonhomologous end joining (NHEJ) or homology-directed (HDR) DNA repair pathways. NHEJ causes random insertions or
deletions (indels) at its targeted site, and HDR can create desired mutations or indels through homologous recombination guided by
donor DNA. A mutation in one nuclease domain of Cas9 creates a Cas9-based nickase (nCas9) that cleaves only one strand of DNA.
The specificity of Cas9-mediated genome editing can be greatly enhanced by using a pair of nCas9s that target each strand of DNA at
adjacent sites because both nCas9–sgRNA complexes must be present at the target site for DSB creation (5, 76–78). A similar strategy
has been achieved by using paired nuclease-deactivated Cas9 (dCas9)-FokI–sgRNA complexes (153, 154).
crRNA repeat sequence is critical for crRNA processing, Cas9 binding, and Cas9-mediated target
cleavage (3, 14). In the third stage, Cas proteins recognize the appropriate target with the guidance
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of the crRNA and mediate the cleavage of the invading genome, thus protecting the host cells from
infection. The action of many CRISPR systems depends on the presence of a sequence-specific
PAM that is adjacent to the crRNA target site in the invading genome (30, 63–65). The absence
of this PAM sequence at the CRISPR locus in the host genome protects it from self-cleavage in
type I and type II CRISPR systems (9).
simple PAM (NGG, or a weaker NAG, where N is any nucleotide; 68) and has been optimized
for use in editing, as well as other contexts, using dCas9 across a wide variety of organisms.
Other Cas9 proteins have been studied and developed as tools. Of note is the Cas9 derived from
Staphylococcus aureus (Sa), which is 1,053 AA in length, with NNGRRT (where R is an A or G) as its
PAM (69). The relatively small size of Sa Cas9 allows it to circumvent some of the delivery issues
caused by the larger Cas9s, such as Sp Cas9 (1,368 AA; see the section Use and Delivery of Cas9
for further discussion). Sa Cas9 has been developed for genome editing (69) and gene regulation
(70) in mammalian cells, and it shows gene editing efficiency comparable to that of Sp Cas9 (69). In
addition to Sp Cas9 and Sa Cas9, other notable orthologs include Cas9 from Neisseria meningitidis
(Nm; PAM = NNNNGATT) and S. thermophilus 1 (St1; PAM = NNAGAAW, where W is an A
or T), which have been used in both bacteria and mammalian cells (71–74). Both Nm Cas9 and
St1 Cas9 have been engineered into dCas9 versions that have been used for gene regulation (38,
39, 71).
Annu. Rev. Biochem. 2016.85:227-264. Downloaded from www.annualreviews.org
In addition to their distinct PAMs, these different Cas9s also have distinct crRNAs and tracr-
RNAs, which allow for the possibility of orthogonal genome editing, regulation, and imaging.
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Cas9–tracrRNA binding is sensitive to minor perturbations in the tracrRNA sequence and struc-
ture (75), which reinforces the orthogonality of these different Cas9s. Although there has been
some work using multiple Cas9s simultaneously to achieve distinct targeting and function (71, 72),
this area remains relatively underexplored.
expands the toolkit of programmable RNA-guided endonucleases for genome editing (57–59).
These newly characterized proteins will no doubt be the source of many tool-building efforts in
the future.
target-strand DNA backbone directly adjacent to the PAM (70, 84). These interactions appear to
kink the DNA and facilitate the local, adenosine triphosphate–independent DNA strand separation
required to initiate the formation of the sgRNA–DNA duplex (70, 84).
Förster resonance energy transfer (FRET) assays, is sensitive to mismatches within the guide
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Proposed Working Model for Guide RNA Binding and Target DNA Cleavage
A working mechanism of Cas9 has been proposed by combining structural studies (70, 81–85) and
in vitro assays (86–88, 91). In this model, the Cas9 protein maintains an autoinhibited conformation
when not bound by sgRNA in which the active sites in the HNH domain are blocked by the RuvC
domain (81). The binding of an sgRNA induces a conformational change to create a central channel
between the two lobes for DNA binding (70, 81–86), thus entering into a DNA recognition–
competent state (82). The resulting Cas9–sgRNA pretargeting complex can survey DNA for PAMs
by three-dimensional diffusion (87, 92). The Cas9–sgRNA complex binds to a PAM through its
PI domain, which initiates local DNA strand separation in the PAM-proximal region to facilitate
sgRNA–DNA heteroduplex formation (84). The Cas9–sgRNA complex will continue to unwind
the DNA only if there is a significant match between the guide RNA segment and the target
DNA (82, 86). The strong guide RNA–target DNA base-pairing interactions further promote
DNA double strand separation and RNA–DNA heteroduplex formation, which proceeds from
the PAM-proximal region and forms a complete R loop (86–88). Finally, the complete R loop
causes another conformational change in the HNH domain, activating the nuclease activity of
both the RuvC and HNH domains to induce DNA cleavage (81–88). Sp Cas9 creates a DSB
approximately 3 nt away from the PAM in the target DNA (3,4).
or mutating a critical region of the encoded protein) (Figure 1b) (93). HDR can be exploited to
generate the desired sequence replacement at the DSB site through homologous recombination
guided by a donor DNA template, causing targeted gene deletion, mutagenesis, insertion, or gene
correction (Figure 1b) (17, 19). Thus, the CRISPR/Cas9 system provides a powerful platform
for sequence-specific genome editing, including gene knockout, gene knockin, and site-specific
sequence mutagenesis and corrections (9, 10, 35).
schemes in a number of models of genetic and infectious diseases (9, 10, 94).
Retargeting Cas9 to a new DNA site is easy to achieve by simply creating a new sgRNA that
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pairs with the desired DNA targeting site adjacent to PAM. In the case of Sp Cas9, the NGG
PAM motif occurs, on average, once every 8 bp within the genome, thus allowing almost any gene
of interest to be targeted (9, 10, 35). Cas9s from other species have different PAMs, of different
sizes and comprising a variety of sequences, which further expands the range of Cas9-targetable
genomic sequence [e.g., Sa Cas9 (69), St1 Cas9, and Nm Cas9 (71)]. The engineering of existing
Cas9s has also led to the creation of new versions of Cas9 with altered PAM sequences (95, 96),
thus expanding the targetable space within the mammalian genome.
The use of the Cas9 platform has greatly increased the efficiency of generating transgenic
organisms, from fungi (32) and plants (33, 34, 97, 98) to a variety of animals (5–7, 36, 99–102)
(reviewed in 9, 10, 35). This technology also makes it much easier to generate disease models
for genetic disorders and diseases such as cancer, which aids our understanding of the molecular
mechanisms of these pathological processes (reviewed in 9, 10, 103).
Cas9 can be easily programmed to edit multiple genomic loci at the same time by introduc-
ing several sgRNAs simultaneously. This can be applied to generate large-scale chromosomal
rearrangements. For example, creating a pair of DSBs at nearby regions within the same chro-
mosome may produce targeted deletions or inversions of the intermediate segment of DNA
(104–110), and creating two DSBs in different chromosomes may lead to a targeted chromosomal
translocation (107, 111). These Cas9-mediated, targeted rearrangements may be useful for creat-
ing disease models by mimicking rearrangements that occur in human disease states (e.g., cancers
and heritable genetic disorders) (107, 110, 111).
The Cas9 system also has the potential to cure or treat many maladies, including HIV, genetic
diseases, and cancer (94, 112). For example, when Cas9 is introduced into infected cells together
with sgRNAs targeting crucial viral genomic elements, it helps to inactivate or clear the viral
genome and, thereby, defends the cells or organism from infections with HIV (113, 114), hepatitis
B virus (115–117), human papillomavirus (118), and Epstein–Barr virus (119). Moreover, it has
been shown by using CRISPR/Cas9 (120, 121) or ZFNs (122–124) that editing the genes of HIV
coreceptors (e.g., CCR5) in the host genome, which encodes a coreceptor of HIV, creates cellular
resistance to the HIV-1 virus and, thereby, may help to combat infection.
In addition, many studies have reported using the Cas9-mediated genome editing system for
correcting disease-related mutations in animal somatic (125) and germ line cells (126–128), as
well as in human stem cells (129) and induced pluripotent stem cells (130–136). A partial list
includes the Fah gene in hereditary tyrosinemia (125), Dystrophin in Duchenne muscular dystrophy
(126, 133), Crygc in cataracts (127, 128), CFTR in cystic fibrosis (129), HBB in β-thalassemia
(132, 134, 135), JAK2 in polycythemia vera (136), and SERPINA1 in α-1 antitrypsin deficiency
(136) (reviewed in 94, 112, 137).
the mature siRNA form by the cell’s endogenous small RNA pathway. In this way, large-scale
gene knockdown screening can be achieved using a library of siRNAs or shRNAs.
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Similarly, by creating a library of sgRNAs targeting gene coding regions, researchers can exploit
the CRISPR/Cas9 platform to screen for genes contributing to a biological process. The Cas9–
sgRNA approach generates indels at the targeted loci and may cause complete loss of gene function,
whereas the RNAi method may lead to only partial gene suppression. Thus, when targeting the
same gene, the CRISPR/Cas9 technique may generate a more pronounced phenotype than RNAi,
which may make identification of relevant genes easier. One avenue to validate candidate genes
identified with the CRISPR/Cas9 approach is to re-express the targeted gene in the knockout
strain (143). Similarly, hits discovered with the RNAi approach may be validated by expression of
an RNAi-resistant transcript (143).
In terms of limitations in targeting, the CRISPR/Cas9 method can target only a sequence ad-
jacent to PAM, and not all exons contain such a targetable sequence, whereas an siRNA or shRNA
library, in principle, can target any mRNA sequence. In addition, a complete gene knockout by
CRISPR/Cas9 requires all alleles of the same gene to be mutated, which makes the screening
more challenging for cells containing several alleles, such as cancer cells (147). Furthermore, use
of the CRISPR/Cas9 knockout approach to study essential genes is challenging, because deletion
of essential genes causes a lethal effect that prevents most functional assays. Both methods can form
the basis of a successful screen, and the method choice will depend on the needs of the experiment.
modulate the HDR:NHEJ ratio, including altering the expression of DNA repair components,
using small molecules, synchronizing the cell cycle, and optimizing delivery timing and methods
(159–163). It is also imperative to develop tightly regulated platforms, as well as safe and efficient
delivery methods, for precise control of Cas9 activity, especially for potential applications in gene
therapy.
gene in human embryos, although this was done in nonviable, triploid zygotes (164). There is
much debate among scientists, bioethicists, policymakers, and the public about how to ethically
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and responsibly use gene editing technology in a way that does not hamper beneficial scientific
research and discovery (165–171). Regarding the editing of human cells, major questions include,
but are not limited to, should editing of human somatic tissues or germ-line cells be allowed and
regulated? Should this technology be used in human embryos? If the answer to these questions is
yes, in which cases would this be applicable?
In addition to the ability to edit the human genome, Cas9 also offers the possibility of drasti-
cally altering ecosystems by editing the genomes of plants and animals. By editing the genomes
of crops and livestock, there is a potential to greatly increase the yield of food production. Cas9-
based genome editing technology also has been proposed as a possible method for controlling the
populations of disease transmitters, such as mosquitoes that transmit malaria (172). This could be
accomplished through the use of gene drive technology, which facilitates the rapid spread of ge-
nomic alterations in wild populations (reviewed in 173). Although there is potential benefit to using
Cas9-based gene drives, there is still much debate about how or if this technology should be used.
Major concerns include doubts about our ability to predict the full ecological impact of such genet-
ically modified organisms and our ability to contain or control them once released into the wild.
The rapid development of Cas9 technology underscores our need as a scientific community
and as a society for a comprehensive policy regarding the use of genome editing technology. As the
rapid pace of biological discovery continues, discussion will be necessary to ensure that genome
editing technologies, such as Cas9, will be used in a safe and responsible manner.
termed CRISPR interference (CRISPRi), as it interferes with the transcription of RNA. Although
CRISPRi is generally highly efficient in prokaryotes, the dCas9–sgRNA complex alone may not
be very efficient at silencing gene expression in mammalian cells (41). However, CRISPRi in
mammalian cells can be enhanced by fusing dCas9 to a transcriptional repressor domain (e.g., the
KRAB domain of Kox1), which leads to successful suppression of reporter and endogenous genes
(Figure 2a) (37, 177).
In addition to CRISPRi, CRISPR activation (CRISPRa) has been created by fusing dCas9
to transcriptional activators, such as VP64 and p65AD in mammalian cells (Figure 2b) (37, 76,
175, 177) and the ω subunit of RNA polymerase in bacteria (176). These dCas9 fusions are
able to upregulate gene expression in host cells. In this review, we focus on the use of CRISPRi
and CRISPRa (CRISPRi/a) in eukaryotic cells. CRISPR/Cas9-based prokaryotic activation and
repression is reviewed in Reference 178.
Annu. Rev. Biochem. 2016.85:227-264. Downloaded from www.annualreviews.org
In addition to direct fusions of an activator or repressor to dCas9, the sgRNA can be modified and
turned into a scaffold to recruit transcriptional regulators (76, 179–181). The sgRNAs can be fused
to orthogonal protein-interacting RNA aptamers, which recruit specific RNA-binding proteins
(RBPs) (Figure 2c). These aptamer-modified sgRNAs are termed scaffold RNAs (scRNAs) (179).
Transcriptional activators and repressors can be fused to these RBPs in lieu of dCas9. When
orthogonal RNA aptamer–RBP pairs (e.g., MS2–MCP, PP7–PCP, com–Com) are coupled to
different sgRNAs, distinct RBP transcriptional modules can be recruited to different genes to
achieve multimodal regulation (i.e., simultaneous activation and repression) (179). For example,
in the presence of Sp dCas9, one gene can be targeted by an scRNA with an aptamer that will
recruit VP64 and cause activation. Another gene can simultaneously be targeted by an scRNA
with an aptamer that will recruit KRAB and cause repression (Figure 2c) (179). Thus, this system
allows for multimodal regulation of different genes within the same cell using a single Sp dCas9
protein (179).
VP64 p65
dCas9
MCP
sgRNA
MS2
RNAP
dCas9-VP64
dCas9 alone (bacteria)
dCas9 SAM system
scRNA
Gene 1 Gene 2
d Epigenetic modification
dCas9
sgRNA ac
me
Gene Gene
Figure 2
Nuclease-deactivated Cas9 (dCas9)-mediated sequence-specific gene regulation. (a) CRISPR interference (CRISPRi) strategies.
Repression can occur with dCas9 alone in bacteria, which sterically blocks transcriptional elongation of RNA polymerases (RNAPs)
(41). Alternatively, dCas9 can be fused to a repressor domain such as KRAB to enhance repression (37). (b) CRISPR activation
(CRISPRa) strategies. Activation can be achieved by directly fusing dCas9 to a transcriptional activator (e.g., VP64) or by recruiting
multiple transcriptional activators using the synergistic activation mediator (SAM), SunTag, or VP64-p65-Rta (VPR) systems (37, 180,
184, 185). Note for the SAM system, each MS2 aptamer can recruit a pair of MCP-p65-HSF1, but only one is shown for simplicity.
(c) Gene activation and repression can occur simultaneously in the same cell using the scaffold RNA (scRNA) system. RNA aptamers
(e.g., MS2, com, PP7) are fused to single guide RNA (sgRNA), creating an scRNA that is localized to a specific genomic locus with
dCas9. The scRNAs can recruit RNA-binding proteins (RBPs; e.g., MCP, Com, PCP) fused to an activator (e.g., VP64, left) or a
repressor (e.g., KRAB, right) (179). (d ) CRISPR-mediated epigenetic modification. The epigenetic landscape can be altered in a
site-specific manner by fusing epigenetic modifying enzymes such as p300 or LSD1 to dCas9. For example, dCas9-LSD1 decreases
H3K4me2 near the targeted enhancer region, resulting in repression of related genes, whereas dCas9-p300 increases H3K27
acetylation at the promoter or enhancer regions and activates the expression of downstream genes (38, 39).
www.annualreviews.org • CRISPR/Cas9 in Genome Editing and Beyond 241
BI85CH10-Qi ARI 9 May 2016 9:29
of multiple copies of VP64 to each dCas9 (Figure 2b) (185). This system has been used to
strongly upregulate chemokine (C-X-C motif) receptor 4 (known as CXCR4), thus enhancing
cell migration in K562 cells. Additionally, Tanenbaum et al. (185) upregulated cyclin-dependent
kinase inhibitor 1B (known as CDKN1B) using the SunTag system, leading to a reduction in cell
growth. The system has also been used for genome-scale gain-of-function screening (40).
In a third strategy, used by Chavez et al. (184), dCas9 was fused to three different activators in
tandem, VP64-p65-Rta (VPR), resulting in a tripartite activator. dCas9-VPR was able to achieve
greater activation of endogenous genes than dCas9-VP64 (Figure 2b). This system has been
used to direct the neuronal differentiation of induced pluripotent stem cells with multiple coex-
pressed sgRNAs (184). All of these studies show that synergistically recruiting multiple activators
to the dCas9 target locus enhances the activation of the CRISPRa system (180, 184, 185). These
engineered systems likely mimic intrinsic cellular gene activation mechanisms, which work by
coordinating the recruitment of multiple activators (186, 187).
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expression of several endogenous genes by targeting the promoter or enhancer regions, and they
showed that the activation was specific using genome-wide RNA sequencing. When targeted to
the HS2 enhancer, dCas9-p300 core increased the level of H3K27 acetylation at both the targeted
enhancer and the promoters of its downstream genes.
The studies (38, 39, 203) described above have demonstrated that dCas9 fusion proteins can
act as sequence-specific, synthetic epigenome modifiers, which not only change local epigenetic
status but also change the gene expression of relevant genes. Given the broad expanse of functional
epigenetic marks—from DNA methylation to histone modifications—future studies are needed
to develop a full toolkit of dCas9-based epigenetic modifiers. Given the possible off-target effects
of dCas9 when using dCas9-mediated epigenome editing systems to target a specific locus, the
specificity and toxicity of such tools should also be assessed.
In the postgenomic era, another challenge for scientists is to understand the correlations between
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the linear genetic information within DNA and its three-dimensional organization within the cell
nucleus. Many studies have revealed that the three-dimensional organization of genomic structure
may play an important part in regulating gene expression and controlling cell differentiation (204–
207). Further research into the correlations between genomic architecture, gene expression, and
cell behavior is hindered by the lack of tools for visualizing sequence-specific genomic dynamics
in living cells. Cas9’s ability to localize to specific sequences within the genome and the ease of
redirecting it to different genomic loci have made it a promising candidate for studying genomic
organization and dynamics in living cells.
Table 1 Comparison of sequence-specific genomic imaging methods: Nucleic acid probes (left), imaging based on protein–DNA interactions (middle), and
CRISPR/dCas9-based genomic imaging (right) have different advantages and disadvantages based on the given application
ARI
244
Labeled nucleotide probe Fluorescent protein (FP) Fluorescent protein (FP)
9 May 2016
dCas9
Wang
sgRNA
Genome
·
9:29
La Russa
·
PNA probe live Native DNA-binding
Qi
Technique ISH imaging LacO/TetO proteins ZF/TALE CRISPR/Cas9
Probe Labeled nucleic acids Labeled PNA probe Lac/Tet repressors-FP Endogenous ZF/TALE-FP dCas9-FPs + sgRNAs
strand (DNA, RNA, DNA-binding
and PNA, etc.) protein-FP
Genomic Any genomic loci Telomeres Genomically integrated Genomic loci containing Repetitive genomic loci Essentially any genomic locus
target LacO or TetO arrays repetitive native (repetitive or nonrepetitive
protein-binding sites sequences)
(telomeres, etc.)
Cells Fixed cells Living cells Living and fixed cells Living or fixed cells (i.e.,
CASFISH)
Advantages Easy probe Enables live Enables live cell imaging Enables live cell imaging
design cell imaging Allows multiple color imaging Easy sgRNA probe design
Allows multiple Multiple color imaging with
color and orthogonal dCas9s or
high-resolution CASFISH
imaging
Disadvantages Restricted to Only shown for Laborious to Restricted to genomic Laborious to Requires multiple sgRNAs to
fixed samples telomere live create and loci that have natural construct many image a nonrepetitive
Lacks dynamic cell imaging characterize sequence-specific ZF/TALE sequence
information Challenges in insertions binding proteins proteins Requires a PAM
delivery Cannot directly So far only
label endogenous restricted to
genomic loci repetitive
sequences
References 208–217 218 219–221 222–224 42–46 47, 72, 185, 225, 226
Abbreviations: Cas9, CRISPR-associated protein 9; FP, fluorescent protein; ISH, in situ hybridization; PAM, protospacer-adjacent motif; PNA, peptide nucleic acid; sgRNA, single guide RNA; TALE, transcription
activator-like effector; ZF, zinc finger.
BI85CH10-Qi ARI 9 May 2016 9:29
some DNA-binding proteins have high binding specificity for cognate DNA sequences in living
cells, and these protein–DNA interactions do not require DNA denaturation.
Initial work to visualize genomic dynamics involved the insertion of Lac/Tet operator tandem
repeats into a specific genomic locus. These exogenously added repeats were then visualized
using their binding proteins fused with FPs (219–221). This method allows us to understand the
dynamics of genomic loci in living cells. However, it is labor intensive to insert repetitive tandem
repeat sequences into a specific genomic locus, and this method cannot be used to directly label
endogenous sequences.
One way to visualize endogenous loci is to co-opt endogenous DNA-binding proteins and
label them with FPs. Some repetitive genomic loci, such as telomeres and centromeres, have na-
tive sequence-specific binding proteins and, thus, can be easily visualized by fusing these binding
proteins to FPs or by immunostaining with related antibodies (222–224). However, the majority
of the human genome sequence lacks unique binding proteins. For this reason, the use of pro-
Annu. Rev. Biochem. 2016.85:227-264. Downloaded from www.annualreviews.org
grammable DNA-binding proteins, such as ZFs, TALEs, and Cas9s, offers a powerful approach
for imaging these genomic loci.
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Several groups have used ZFs or TALEs to image endogenous genomic loci. Initial efforts were
made by fusing green fluorescent protein (GFP) to ZF proteins to image repetitive sequences
at pericentric regions in living cells (42). Several studies (43–46) have also imaged repetitive
genomic elements by fusing FPs to TALEs. Miyanari et al. (43) reported a TALE-mediated
genome visualization method used to track the dynamics of centromeres and telomeres in living
mESCs and mouse embryos, and they also used this method to efficiently distinguish two parental
chromosomes with distinct single nucleotide polymorphisms, suggesting a high specificity. Ma
et al. (44) published similar and complementary results showing that two TALEs tagged with
different colors can simultaneously track the dynamics of centrosomes and telomeres in living
cells. They also showed in vitro purified TALE proteins could label genomic loci in fixed cells by
using a protocol simpler than FISH. Another work by Thanisch et al. (45) used a FP-TALE to
track the dynamics of satellite repeats throughout the cell cycle in mESCs.
loci separated by around 2 mega bases (72). Because the three dCas9s recognize distinct sgRNAs
and PAM sequences, this system provides a tool for tracking the dynamics of multiple genomic
loci simultaneously.
In a method termed CASFISH, fixed cells and tissues can also be efficiently labeled by fluo-
rescently labeled dCas9–sgRNA complexes assembled in vitro (226). Compared with traditional
FISH, which uses high temperature and formamide for denaturation prior to probe hybridization,
CASFISH allows rapid sequence-specific labeling using the dCas9-mediated enzymatic reactions
and, thus, may help to preserve cellular and genomic structure. The authors were able to image
repetitive sequences with a single sgRNA and to image nonrepetitive sequences with an array
of sgRNAs. Given that preassembled Cas9–sgRNA complexes are stable, the authors were also
able to demonstrate multicolor imaging using separately preassembled Cas9–sgRNA complexes
targeting different loci with different colors.
Visualizing the dynamics of fluorescently labeled dCas9 also provides insights into the mecha-
Annu. Rev. Biochem. 2016.85:227-264. Downloaded from www.annualreviews.org
nism of how Cas9 searches the genome for its target sites in living cells. Fusing dCas9 to a HaloTag
system and using a single-particle tracking method, Knight et al. (92) found that dCas9 mainly
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surveys the genome through three-dimensional diffusion, with transient off-target binding. They
also found that dCas9 on-target binding lasts much longer than off-target binding and observed
reduced searching efficiency in heterochromatic regions.
necessary to use additional assays to validate the results from CRISPR enChIP.
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Split Cas9s
In addition to light-inducible two-component systems, different versions of split Cas9 have been
created to allow inducible control of gene editing and regulation. For example, Zetsche et al. (233)
split Cas9 into N-terminal and C-terminal fragments and used a rapamycin-binding dimerization
system to induce fragment assembly into active Cas9. Due to their autoassembly, the two split Cas9
fragments need to be spatially separated by tagging with, respectively, nuclear export sequences and
nuclear localization sequences. These systems then make use of rapamycin-induced dimerization
to shift the whole complex to the nucleus for DNA targeting. In addition to genomic editing,
inducible gene activation is also achieved with this system by fusing split dCas9 to VP64. A similar
approach has been used to construct split Sa Cas9 for inducible genomic editing (70). Another
strategy reported by Wright et al. (234) uses a split Cas9 that is separated into a nuclease lobe
peptide and an α-helical lobe peptide. An sgRNA is sufficient to induce their assembly into a
whole Cas9 nuclease, although the efficiency of the split Cas9 for genome editing is much lower
than for wild-type Cas9.
Nihongaki et al. (235) created a photoactivatable Cas9 system by fusing split Cas9 fragments
(N713 and C714) with light-inducible dimerization domains. Blue-light-induced dimerization
allowed split Cas9 fragments to reconstitute nuclease activity. This system also allows for optoge-
netic control of nCas9 activity as well as dCas9-mediated transcriptional repression. By tuning the
region and timing of blue light excitation, spatiotemporal and reversible control of Cas9 nuclease
Annu. Rev. Biochem. 2016.85:227-264. Downloaded from www.annualreviews.org
vivo delivery of Cas9. Cas9 was split into N-terminal and C-terminal fragments that were fused
to each of the two components of intein, respectively. The two fragments of Cas9-intein fusions
can be delivered into cells in separate vectors. After intein self-splicing, a full-length Cas9 forms,
exhibiting comparable gene targeting efficiency to wild-type Cas9 in cells. This method allows
the large Sp Cas9 to easily fit into the packaging limit of the recombinant adeno-associated virus
(AAV) system, although its in vivo effectiveness still needs to be assessed.
The development of split Cas9 systems provides new strategies for regulating Cas9 activity,
and it may facilitate the delivery of Cas9 by bypassing the size limitations of some delivery systems.
However, with the exception of intein-mediated split Cas9 (236), most split Cas9 systems have
shown reduced nuclease activity compared with full-length Cas9 (233–235). Additionally, the
background activity of split Cas9 due to autoassociation needs to be critically evaluated.
The exciting potential of using Cas9 to target RNA may inspire further development of Cas9-
based tools for RNA manipulation. For example, by coupling RNA-targeting Cas9 to different
modulators, it may be possible to regulate the stability, localization, and splicing of the targeted
RNA and to track the real-time dynamics of RNA processing (239).
to 3 or more out of 6 sgRNAs for 90% of genes. Gilbert et al. (40) used a similar technique to
define the rules for effective CRISPRi/a sgRNA, tiling thousands of sgRNAs in a 10 kb window
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around 49 genes. They defined a set of rules from which it is possible to obtain at least 1–2 highly
active (80–99% repression) sgRNAs out of a pool of 5–10. Currently, it is usually necessary to
clone a handful of sgRNAs per target gene, as there are other factors affecting sgRNA function
that have not yet been defined.
Although there are several online tools available to aid in designing sgRNAs, it is also possible
to design sgRNAs manually. Several groups have distilled information from large data sets to come
up with a set of simple rules for what makes an sgRNA most effective in various contexts. Design
rules include, for example, the following:
All sgRNAs must be adjacent to a PAM site: Sp Cas9 uses NGG or a less efficient NAG
(3, 68). Sa Cas9’s PAM is NNGRRT (69).
When using a U6 promoter for sgRNA expression in mammalian cells, the first nucleotide
of the sgRNA must be a G for effective expression, although it has been suggested that an A
may work in some contexts (240). It has been shown with Cas9 nuclease that an sgRNA can
still function if the 5 G of the sgRNA is mismatched with the target site, which is useful if
no target site can be found where a G is 18–25 bp upstream of NGG (139, 241).
An sgRNA expressed from a U6 promoter in mammalian cells should not contain a stretch
of four or more uracils (U’s) in a row or it will be terminated prematurely due to the activity
of RNA polymerase III (242). A stretch of U’s near the 3 end of the guide sequence is
unfavorable for Cas9–sgRNA binding (138).
There are mixed reports about the effect of GC content. An article by Wang et al. (138) sug-
gested that sgRNAs with a very high or low GC content were less effective when combined
with nuclease Cas9. Another study, by Gilbert et al. (40), used dCas9 fused to effectors and
found that variations in GC content did not significantly change sgRNA effectiveness.
Long stretches of the same nucleotide greatly decrease sgRNA activity (40).
Although great strides have been made in finding ever better sgRNAs, the design rules for both
Cas9-based genome editing and gene regulation will no doubt benefit from further optimization
in the future.
a frameshift mutation (138, 139). To determine the most potent targeting sites for CRISPRi/a,
Gilbert et al. (40) and Konermann et al. (180) have tiled sgRNAs around the promoters of various
genes. By tiling a library of sgRNAs in a 10 kb window around the TSS of 49 genes (approximately
55,000 sgRNAs total), Gilbert et al. (40) systematically examined which sites allowed for the most
active sgRNAs for both CRISPRa and CRISPRi. CRISPRi is most effective with sgRNAs in
the −50 to +300 bp window around the TSS, with the absolute highest repression occurring in
approximately the 50–100 bp window downstream of the TSS. The most effective sgRNAs for
CRISPRa are targeted to a window −400 to −50 bp upstream of the TSS. Both of these activity
windows are consistent with data regarding the mechanism of action for the KRAB repressor (243,
244) and VP64 activator (245).
Although these studies have been greatly informative for the creation of effective sgRNAs,
much work still remains for figuring out the full set of rules, particularly for different cell types
and organisms. Future work should systematically determine how local chromatin, transcriptional,
Annu. Rev. Biochem. 2016.85:227-264. Downloaded from www.annualreviews.org
and epigenetic status (i.e., local histone marks, methylated DNA, or actively transcribing DNA)
affect genome targeting, binding, nuclease, and regulatory activities. For example, there is a positive
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correlation between the level of sgRNA expression in mammalian cells and the regulatory function
of Cas9, suggesting that the sgRNA expression level is likely a limiting factor for the dCas9 function
(7). For CRISPRi/a, the absolute dosage of sgRNAs and dCas9, as well as the most effective ratio
of the two, also require future study.
Delivery Methods
There are many vectors available on Addgene (https://www.addgene.org/) and through com-
mercial vendors that encode various CRISPR components. The particular components and their
delivery will depend on the assay and cell type. After picking an appropriate Cas9-encoding vector,
a complementary sgRNA-encoding vector can also be adopted. Directing a dCas9 effector to a
sequence of interest requires the simple cloning of a short sgRNA (approximately 100 bp) and
then introducing both components into the cell type of interest (141, 174, 188). For CRISPRi/a, it
is standard to also use one or multiple nontargeting sgRNAs—which should not target anywhere
in the genome or introduced DNA—as a negative control and as a way to normalize results.
Depending on the desired application, there are various ways in which the Cas9 system can
be introduced. For an application such as a pooled screen, it is often desirable to make a stable
Cas9 or dCas9 effector cell line using lentiviral or retroviral vectors before the introduction of a
lentiviral sgRNA pool (138). This method may be problematic when working with primary cells.
Alternatively, it is possible to use vectors that encode both Cas9 and a single sgRNA (139, 141).
It is necessary to have just one sgRNA present per cell in pooled screens because this gives the
highest signal-to-noise ratio when correlating an observed phenotype with the effect of a given
sgRNA. Thus, viral delivery is ideal for pooled screens because the viral particles can be titered
such that, on average, there is less than one sgRNA-containing viral particle per cell in the infected
pool (giving a multiplicity of infection of less than 1).
For shorter-term or smaller-scale experiments in cell lines, it is also possible to use transient
transfection to introduce plasmids containing Cas9 or dCas9 effectors and sgRNAs. When knock-
ing out or editing a gene or handful of genes, this may be ideal, as continued expression of the
CRISPR components is not necessary once the desired genome modification has occurred. For
CRISPRi/a, transfection is best suited to activation assays, which can often be assessed after 24–
48 hours (37). Repression assays may be less effective when using transfection because CRISPRi
affects transcriptional efficiency and not mRNA or protein stability. The already-present mRNA
and protein for the target gene must be degraded at its normal turnover rate, which can vary from
gene to gene, before the full effects of repression are seen. It may take several days to see repression,
but by that time the CRISPRi plasmids may have been diluted out of the cells as they divide and,
thus, CRISPRi may become less effective. Thus, any studies using CRISPRi may be better suited
to viral delivery to maintain CRISPRi component expression over a longer time scale. With an
eye toward the technical aspects of Cas9–sgRNA component expression and delivery, it is quite
straightforward to design an effective assay.
For gene therapy, the delivery of the Cas9 system is more challenging and raises more safety
concerns. AAV-based vectors are the preferred candidates for somatic gene therapy due to their
mild immune response, lack of pathogenicity, and ability to target nondividing cells (246). How-
ever, the coding sequences of Sp Cas9, the most widely used Cas9, and its sgRNA are already
approaching the packaging limit of AAV-based gene therapy vectors. The large size of Sp Cas9
makes its usage in AAV-based gene therapy unfavorable, especially when promoter sequences,
localization signals, donor DNA, or additional sgRNAs are needed. The Sa Cas9 has an ap-
Annu. Rev. Biochem. 2016.85:227-264. Downloaded from www.annualreviews.org
proximately 1 kb shorter coding sequence than Sp Cas9, allowing it to be easily packaged into
AAV-based vectors for gene editing (69, 247). Using this AAV Sa Cas9 approach, Ran et al. (69)
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efficiently induced indel formation of more than 40% of the cholesterol regulatory Pcsk9 (propro-
tein convertase subtilisin/kexin type 9) gene in mouse liver after 1 week of treatment, causing a
95% drop in serum Pcsk9 and a 40% drop in total cholesterol.
Besides viral systems, nonviral methods have been used to deliver Cas9-encoding plasmids or
Cas9 mRNA into animal cells and tissues. For example, coinjection of Cas9-encoding mRNA and
an sgRNA into zygotes or embryos can generate genome-edited animals (36, 128, 248, 249). Cas9-
containing vectors can also be introduced into adult animals through hydrodynamic injections,
leading to efficient gene corrections or mutations (125, 250).
The use of purified Cas9–sgRNA ribonucleoproteins (RNPs) is another option for cellular
delivery. Compared with a viral or nonviral nucleotide delivery method, the RNP delivery systems
allows for fast action of the RNP complex in the nucleus and a shorter duration of the Cas9
nuclease presence in the cells and, thus, it may increase the efficiency and reduce the off-target
effects. RNP delivery also avoids undesired genomic alterations that can occur when using other
nucleotide delivery methods.
RNP delivery of Cas9 and sgRNAs can be achieved through a variety of strategies. Many
methods traditionally used for nucleotide transfection have been proven useful for Cas9 RNP
delivery, including microinjections, electroporation, and lipid-mediated transfection. Microinjec-
tion of purified Cas9 RNP complexes into animal embryos successfully generated genome-edited
animals (251, 252). Electroporation methods have been established to introduce Cas9–sgRNA
complexes into primary cells and embryonic stem cells, and these can induce targeted gene muta-
tions and large chromosome deletions while minimizing the off-target effects (108, 161, 253–256).
Commercially available nucleotide transfection reagents have also been adapted to deliver Cas9–
sgRNA RNP complexes (256, 257). Given that the Cas9–sgRNA complex is inherently anionic,
Zuris et al. (257) adapted cationic lipid transfection reagents to deliver the Cas9 nuclease, nCas9,
and the dCas9-VP64 transcriptional activator. This method allowed for gene modification of up
to 80% in cultured cells and approximately 20% in mouse inner ear hair cells in vivo (257).
Other approaches have also been explored for delivering RNP complexes. For example,
Ramakrishna et al. (258) used cell-penetrating peptides (CPPs) to induce gene editing through
Cas9 conjugated with CPPs and through the sgRNA in complex with CPPs. These CPP com-
plexes entered cells in the form of positively charged nanoparticles to achieve gene editing in a
variety of human cell types. D’Astolfo et al. (259) developed a method of induced transduction by
osmocytosis and propanebetaine (termed iTOP) to deliver Cas9–sgRNA complexes, leading to
highly efficient gene editing in primary cells. This method allows uptake and release of proteins
d Genomic imaging
Fluorescent protein Imaging of repetitive/
nonrepetitive genomic
loci targeted by dCas9
Live/fixed cell imaging
and other molecules in a variety of primary cells through the macropinocytosis pathway. In addi-
tion, Sun et al. (260) made use of a DNA nanoparticle, termed a nanoclew, to deliver a Cas9 RNP
complex into U2OS cells for gene editing. They also used nanoclews to deliver the Cas9–sgRNA
complex in vivo, successfully editing a genome-integrated GFP reporter in 25% of U2OS tumor
cells, which had been xenografted in mice.
Compared with vector-mediated nucleotide delivery methods, Cas9-mediated genome editing
via RNP delivery methods (108, 161, 253, 257, 258) exhibits higher fidelity and lower cell toxicity,
bypasses the safety problems of introducing foreign DNA into the host genome and, thus, may
provide a platform for developing gene therapy tools.
brought forth revolutionary changes in genomic research, including genome editing, regulation,
and imaging (Figure 3). As we look to the future, we can envision the advances that CRISPR/Cas9
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technology will bring to basic bioscience researchers and clinicians. Basic scientists are already
making great strides in understanding and manipulating biology using CRISPR/Cas9 technology.
In the clinic, we can look forward to new therapies for genetic diseases (using Cas9 genome editing
or CRISPRi/a) and new diagnostic techniques (using dCas9-based imaging).
Basic research similar to that which uncovered CRISPR/Cas9 allows us to make use of nature’s
technological toolbox, which has been honed for billions of years through evolution. Although
most research thus far has focused on the type II Cas9 proteins, there is much still to be discovered
about the broader CRISPR systems from other diverse species of bacteria and Archaea. New
CRISPR systems, hidden in plain sight in the genomes of the organisms around us, may continue
to surprise us in the future with their elegant mechanisms and function, offering powerful and
groundbreaking technologies.
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
The authors thank all the members of the Qi lab, as well as Dr. Fuguo Jiang and Arjun Aditham
for advice and helpful discussions. Thanks also to Matias Kaplan for preparing the structure
comparison of Cas9s. L.S.Q. acknowledges support from National Institutes for Health’s Office
of the Director and the National Institute of Dental and Craniofacial Research. M.L. acknowledges
support from an Agilent University Relations grant. This work was supported by NIH grants R01
DA036858 and U01 EB021240 (H.W., L.S.Q.) and DP5 OD017887 (M.L., L.S.Q.).
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